Swine vesicular disease virus (SVDV) is an enterovirus of the picornaviridae family and causes swine vesicular disease. The mature virus particle contains (+)-strand polyadenylated RNA generally about 7,400 bases in length. Thus, the SVDV genome is also a functional mRNA, which is composed of P-1, P-2, and P-3 regions (FIG. 1). The entire viral genome contains only one open reading frame, from which a precursor polyprotein is generated. The polyprotein is cleaved into mature viral proteins by a virus-specific protease (see P2 region in FIG. 1). The P-1 region contains four viral structural proteins, VP-1, VP-2, VP-3, and VP-4 (also known as 1A, 1B, 1C, and 1D, respectively), which form the capsid of the virus. The P-2 region contains non-structural viral proteins 2A, 2B, and 2C; and the P-3 region contains non-structural proteins 3A, 3B, 3C, and 3D. As used herein, the P-1, P-2, and P-3 regions can refer to either the nucleotide sequences in these regions or the amino acid sequences encoded thereby.
During the virus life cycle, SVDV enters the cell, releases the genome into the cell, and translates viral proteins from the genomic mRNA. Using the viral RNA-dependent RNA polymerase, the virus produces (xe2x88x92)-strand RNA, which in turn serves as a template for the production of more (+)-strand RNA for translation or packaging into virions. Further details regarding SVDV virology can be found in Kanno et al., J. Virol. 73:2710-2716, 1999; Zhang et al., J. Gen. Virol. 80:639-651, 1999; and references cited therein.
The invention is based on the discovery that cloned SVDV genomes, in both RNA and DNA form, can be used as vectors for expressing heterologous RNA or protein. Particularly, it has been discovered that replacement of the P1 region of a SVDV genome with a relatively large heterologous nucleotide sequence is sufficient for expression of heterologous RNA and protein.
Accordingly, the invention features a nucleic acid (i.e., an RNA or DNA molecule) having a nucleotide sequence encoding a polypeptide, the polypeptide containing (1) the P2 and P3 regions of a swine vesicular disease virus polyprotein, and (2) a heterologous amino acid sequence.
A heterologous amino acid sequence can be 8, 10, 20, 50, 100, or 200 or more amino acids in length. In addition, a heterologous nucleotide sequence can be 22, 50, 100, 200, or 400 or more bases or base pairs in length. The term xe2x80x9cheterologousxe2x80x9d means that the amino acid or nucleotide sequence does not fully correspond to a genomic or polyprotein sequence of a naturally occurring SVDV. A xe2x80x9cpolyproteinxe2x80x9d is the polypeptide produced by translation of a SVDV genome (e.g., a naturally occurring SVDV genome).
The nucleic acids of the invention can further include a promoter operably linked to the nucleotide sequence, i.e., the promoter is configured to transcribe an RNA encoded by the nucleotide sequence.
The invention also includes (1) an RNA molecule having a swine vesicular disease virus genome and a heterologous nucleotide sequence inserted into or replacing the P1 region of the genome, and (2) a swine vesicular disease virus whose genome contains a heterologous nucleotide sequence inserted into or replacing the P1 region of the genome.
Also included in the invention is a method of producing a swine vesicular disease virus expression vector by providing a DNA molecule having (1) a nucleotide sequence encoding the P2 and P3 regions of a swine vesicular disease virus polyprotein, and (2) a promoter operably linked to the nucleotide sequence; and inserting a heterologous nucleotide sequence into the DNA molecule, where transcription from the promoter results in an RNA comprising the nucleotide sequence encoding (a) the P2 and P3 regions and (b) the heterologous nucleotide sequence. The heterologous nucleotide sequence can encode a heterologous amino acid sequence, and the RNA can encode a fusion protein containing (1) the P2 and P3 regions and (2) the heterologous amino acid sequence. The method can further include transcribing (in vitro or in vivo) the RNA from the promoter to produce an RNA expression vector.
The invention also includes a kit for expression of a heterologous protein, the kit containing a nucleic acid having (1) a nucleotide sequence encoding the P2 and P3 regions of a swine vesicular disease virus polyprotein, and (2) a polylinker flanking the nucleotide sequence and configured for insertion of a heterologous nucleotide sequence. A xe2x80x9cpolylinkerxe2x80x9d is a nucleotide sequence having artificially introduced restriction endonuclease recognition sites, especially sites of five or more base pairs in length. A polylinker can be 10, 20, 50, or even 100 or more nucleotides in length, and can have a concentration of restriction endonuclease recognition sites of at least two per 10 base pairs, on average, for the entire length of the polylinker. The nucleic acid can further include a promoter operably linked to the nucleotide sequence, and the kit can further include a cell susceptible to infection by a SVDV.
Additionally, the invention features a method of expressing an RNA in a cell by introducing into a cell (e.g., MVPK porcine kidney cells) a DNA molecule having (1) a DNA sequence encoding an RNA molecule including (a) a nucleotide sequence encoding the P2 and P3 regions of a swine vesicular disease virus polyprotein, and (b) a heterologous nucleotide sequence, and (2) a promoter operably linked to the DNA sequence; and culturing the cell under conditions sufficient to transcribe the RNA from the promoter. Where the promoter is inducible, the cell is generally contacted with an inducer compound in a culture to transcribe the RNA. If no inducer is required for expression, standard tissue culture conditions are sufficient for expression of the RNA. As described above, the heterologous nucleotide sequence can encode a heterologous amino acid sequence.
If expression of fusion protein is desired, the method of expressing an RNA can include the additional step of translating the RNA to express a fusion protein having (1) the P2 and P3 regions and (2) the heterologous amino acid sequence. In many cases, only continued culturing of the cell is required for translation.
Also featured in the invention is a composition (e.g., a vaccine composition) containing a nucleic acid of the invention. Where the composition is intended to be a vaccine, the heterologous amino acid sequence would encode an antigen, such as a SVDV antigen, to produce a SVDV vaccine. Other antigens can include tumor antigens, viral antigens (e.g., that of HCV), bacterial antigens (e.g., that of a pathogenic Escherichia coli), and antigens of eukaryotic pathogens (e.g., of a yeast, mycoplasma, protist, fungus, or worm).
The nucleic acids and methods of the invention can be used as a new platform technology for expressing RNA and polypeptides. The relatively simple genomic structure and virus life cycle of SVDV allows for easy manipulation of genetic material and introduction of such material into cells. An exemplary SVDV suitable for use in the invention is the Taiwan Yu-Li strain, which is described in U.S. patent application Ser. No. 09/116,032, now allowed.
Other features or advantages of the present invention will be apparent from the following detailed description, and also from the claims.